The present invention relates to tetraether lipid derivatives, to liposomes and lipid agglomerates containing the inventive tetraether lipid derivatives, and to the use thereof.
Scientific research work requires a great number of methods for the transfection of cells in cell culture and multicellular organisms with nucleic acids. Conventional methods, such as electroporation, DEAE and "calcium phosphate"-supported transfection, microinjection or ballistic methods, have the drawback that the transfection efficiencies achieved by them are often poor, that the cell survival rates are very small and/or that they cannot be performed on multicellular organisms. Although viral and retroviral transfection systems are more efficient, they have risks of their own, such as an increased immune response or an uncontrolled integration into the target genome. Therefore, transfection of non-viral nucleic acids with the aid of liposomes, also called lipofection, is a successful and frequently employed alternative to the above-described methods.
Liposomes are artificially produced unilamellar or multilamellar lipid vesicles which enclose an aqueous interior. They are in general similar to biological membranes and, therefore, they are often easily integrated into the membrane structure after attachment to the membranes. During this membrane fusion the contents of the liposome interior is discharged into the lumen which is enclosed by the biological membrane. Alternatively, the liposomes are moved by endocytotic processes into the cytosol of the cell to be transfected; subsequently they are either destroyed in the cytosol or they interact as such with the nuclear membrane. In the last-mentioned case the compounds contained in the aqueous interior of the liposome are substantially protected against proteolytic or nucleolytic attacks.
Therefore, liposomes can be used as transport vehicles for substances, such as nucleic acids or pharmaceuticals. For instance, the cosmetic industry produces liposome-containing creams for skin care which transport active agents in a target-directed manner into the epidermis or lower cell layers. Natural lecithins of soybean or egg yolk and defined natural or artificial phospholipids, such as cardiolipin, sphingomyelin, lysolecithin, and others, are mainly used for the preparation of liposomes. Size, stability and absorbency as well as the release of the associated molecules are influenced by varying the polar head groups (choline, ethanolamine, serine, glycerine, inosite), the length as well as the saturation degrees of the hycrocarbon atom chains.
One of the essential drawbacks of the liposomes thus far known is their low stability. Even in a cooled state liposomes which are formed from normal bilayer-forming phospholipids are only durable for a short period of time. Although their storage stability can be increased e.g. by the inclusion of phosphatidic acid, the stability improved thereby is still inadequate for many purposes. Moreover, conventional liposomes are not acid-stable and are thus neither suited for the transport of pharmaceutical active agents which pass through the stomach after oral administration, nor for the liposome-supported DNA transfection under slightly acid pH conditions.
Liposome-forming lipid mixtures, such as Lipofectamin.RTM., Lipofectin.RTM. or DOTAP.RTM., are often used in mammalian cells for scientific or medicinal lipofections. Apart from the already mentioned drawbacks, their use requires an exact determination of a multitude of parameters (such as cell density, amount of nucleic acid, amount of the lipids, volume of the liposome batch, etc.) because there is only a very limited range of optimum parameters within which an adequate transfection efficiency can be achieved. As a result, transfections using commercial lipofection reagents become very troublesome and expensive. Furthermore, great variations between the individual charges can be observed in the above-mentioned products, which makes them hardly reliable in practice.